Agricultural Microbiology

Unit 4Biopesticides

B.Sc Agriculture II

Many microorganisms are attracted by nutrients exudedfrom plant roots and this “rhizosphere effect” was firstdescribed by Hiltner. He observed higher numbers andactivity of microorganisms in the vicinity of plant roots.

The rhizosphere and rhizoplane are colonized moreintensively by microorganisms than the other regions ofthe soil. Some of these microorganisms not only benefitedfrom the nutrients secreted by the plant roots but alsobeneficially influence the plants, resulting in a stimulationof their growth.

For instance, rhizobacteria can fix atmospheric nitrogen,which is subsequently used by the plants, therebyimproving plant growth in the soil deficient of nitrogen.

Other rhizobacteria can directly promote the plant growthby the production of hormones. These rhizobacteriapositively influence plant growth and health and oftenreferred as plant growth promoting rhizobacteria (PGPR).

However, their effects are complex and cumulative becauseof in teractions of plants, pathogens, antagonists, andenvironmental factors .

Genera of PGPR include

Azotobacter, , Azospirillum , Pseudomonas ,Acetobacter , Burkholderia , Bacillus, Paenibacillus,and some are members of the Enterobacteriaceae.

Direct use of microorganisms to promote plantgrowth and to control plant pests continues to be anarea of rapidly expanding research.

Rhizosphere colonization is one of the first steps in thepathogenesis of soil borne microorganisms. It is alsocrucial for the microbial inoculants used asbiofertilizers, biocontrol agents, phytostimulators,and bioremediators.

Pseudomonas spp. are often used as model root-colonizing bacteria.

The beneficial effects of these rhizobacteria have beenvariously attributed to their ability to produce variouscompounds including phytohormones, organic acids,siderophores, fixation of atmospheric nitrogen,phosphate solubilization, antibiotics and some otherunidentified mechanisms.

Motile rhizobacteria may colonize the rhizospheremore profusely than the non-motile organismsresulting in better rhizosphere activity and nutrienttransformation.

They also eliminate deleterious rhizobacteria from therhizosphere by niche exclusion thereby better plantgrowth.

Induced systemic resistance has been reported to beone of the mechanisms by which PGPR control plantdiseases through the manipulation of the host plant’sphysical and biochemical properties.

PGPR are beneficial for plant growth and also referredas yield increasing bacteria (YIB). They can affect plantgrowth and yield in a number of ways andenhancement of vegetative and reproductive growth isdocumented in a range of crops like cereals, pulses,ornamentals, vegetables, plantation crops and sometrees.

Treatments with PGPR increase germinationpercentage, seedling vigor, emergence, plant stand,root and shoot growth, total biomass of the plants,seed weight, early flowering, grains, fodder and fruityields etc.

Though the exact mechanisms involved in growthpromotion are still unclear, various mechanisms havebeen suggested to explain the phenomenon of plantgrowth promotion.

These include increase in the nitrogen fixation, productionof auxins , gibberellins, cytokinins, ethylene, solubilizationof phosphorous, oxidation of sulfur, increase in availabilityof nitrate, extra cellular production of antibiotics, lyticenzymes, hydrocyanic acid, increases in root permeability,strict competition for the available nutrients and root sites,suppression of deleterious rhizobacteria, and enhancementin the uptake of essential plant nutrients etc.

However, experimental evidence suggests that bacterially-mediated phytohormone production is the most likelyexplanation for PGPR activity in the absence of pathogens.

while siderophore production by PGPR may be importantfor plants growth stimulation when other potentiallydeleterious rhizosphere microorganisms are present in therhizosphere

Biocontrol Plant pathogens such as fungi, bacteria,viruses, nematodes etc., which cause various diseasesin crop plants are controlled by PGPR.

Mechanisms of biocontrol may be competition orantagonisms; however, the most studied phenomenonis the induction of systemic resistance by theserhizobacteria in the host plant.

PGPR control the damage to plants from pathogens bya number of mechanisms including: out-competingthe pathogen by physical displacement, secretion o

siderophores to prevent pathogens in the immediatevicinity from proliferating, synthesis of antibiotics andvariety of small molecules that inhibit pathogen growth,production of enzymes that inhibit the pathogen andstimulation of the systemic resistance in the plants. PGPRmay also stimulate the production of biochemicalcompounds associated with host defense.

Enhanced resistance may be due to massive accumulationof phytoalexins, phenolic compounds, increase s in theactivities of PR-proteins, defense enzymes and transcripts,and enhanced lignification.

Biocontrol may also be improved by genetically engineeredPGPR to over express one or more of these traits so thatstrains with several different anti-pathogen traits can actsynergistically .

Rhizobacteria-mediated ISR has been reported to beeffective against fungi, bacteria and viruses, but appears toinvolve different signaling pathways and mechanisms.

uctural mechanisms PGPR can induce structuralchang es in the host and these changes werecharacterized by a considerable enlargement of thecallose-enriched wall appositions deposited onto theinner surface of cell wall in the epidermis and outercortex, callose deposition and lignification.

A strain of Pseudomonas fluorescens functions as anactivator of plant disease resistance by inducingcallose synthesis in tomato.

Bean roots bacterized with a saprophytic fluorescentpseudomonad, had higher lignin content than control

Treatment of PGPR significantly reduced germination ofsporangia and zoospores of Phytophthora infestans on theleaf surface of tomato than the leaves of the non-inducedcontrol. Serratia plymuthica strain R1GC4 sensitizessusceptible cucumber plants to react more rapidly andefficiently against Pythium ultimum attack through theformation of physical and chemical barriers at sites offungal entry.

Pseudomonas fluorescens induced accumulation of ligninin pea roots. Bacillus pumilus SE34 showed a rapidcolonization of all tissues including the vascular stele intomato and induced resistance against Fusariumoxysporum.

The main facets of the altered host metabolism concernedthe induction of a structural response at sites of fungalentry and the abnormal accumulation of electron-densesubstances in the colonized areas.

Biochemical mechanisms PGPR are known to produceantibiotics, antifungal metabolites, enzymes, phenolics,signal compounds and other determinants of defense inresponse to pathogen attack. Various antibiotics likebacilysin, iturin-like lipopeptides, diacetylphloroglucinoland pyrrolnitrin, HCN, phenazine-1-carboxylate areproduced by rhizobacteri.

Rhizosphere colonization by Pseudomonas aeruginosa7NSK2 activated phenlyalanine ammonia lyase (PAL) inbean roots and increased the salicylic acid levels in leaves.Increased activity of PAL was observed in P. fluorescenstreated tomato and pepper plants in response to infectionby F. oxysporum f. sp. Lycopersici and Colletotrichumcapsici.

In bean, rhizosphere colonization of various bacteriainduced peroxidase (PO) activity, The higher PO activitywas noticed in cucumber roots treated with Pseudomonascorrugata and inoculated with Pythium aphanidermatum.

Foliar application of P. fluorescens increased chitinase andglucanase activities in rice . Groundnut plants, sprayedwith P. fluorescens strain Pf1, showed significant increasein activities of PAL, phenolic contents, chitinase andglucanase 23-kDa thaumatin-like protein (TLP) and a 30-kDa glucanase. Earlier and increased activities ofphenylalanine ammonia lyase (PAL), peroxidase (PO) andpolyphenol oxidase (PPO) were observed in P. fluorescensPf1 pretreated tomato and hot pepper plants challengedwith P. aphanidermatum . Phenolic compounds are toxic topathogens in nature and may increase the mechanicalstrength of the host cell wall.

Accumulation of phenolics by prior application of P.fluorescens in pea has been reported against P. ultimumand F. oxysporum f. sp. pisi.

Similarly, Serratia plymuthica induced the accumulation ofphenolics in cucumber roots following infection by P.ultimum.

Moreover, PGPR-mediated ISR triggered thehypersensitive reaction (HR), causing death ofindividual cell of leaves following inoculation with thepathogen.

Analysis of H2 O2 content, showed that H2 O2increased significantly in all treatments 12 h afterpathogen inoculation, compared to non-inducedcontrol.

Mechanisms of rhizobacteria-media ted induced systemicresistance (ISR) to the large extent are unknown. ISR inArabidopsis mediated by rhizobacteria is not associatedwith a direct effect on expression of known defense-relatedgenes but stimulated the expression of the jasmonate-inducible gene Atvsp upon challenge. Gene expressionstudies were performed with Arabidopsis gene-specificprobes for the defense-related genes PR-1., PR-2., PR-5.,Hel ChiB ,Atvsp Lox1, Lox2, Pal1 , and Pin2.

Responsiveness of genes to the defense signaling moleculesSA, ethylene, and jasmonate was verified by analyzing theirexpression in leaves treated with SA, the ethylene precursor1-aminocyclopropane-1-carboxylate (ACC), or methyljasmonate ( MeJA). Although variation in the expression ofmost genes was apparent, roots and leaves of P. fluorescensWCS417r-treated plants never showed an enhancedexpression of any of the genes, at any time tested

PPO transcript levels increased in young leaves of tomatowhen mature leaflets were injured (Thipyapong andSteffens, 1997). Increase in mRNAs encoding PAL andchalcone synthase were recorded in the early stages of theinteraction between bean roots and various rhizobacteria .ISR in A . Thaliana by P. fluorescens WCS417r andsubsequent inoculation of Pseudomonas syringae pv.

Tomato Dc3000(ISR) functions independently of salicylicacid but requires an intact response to the plant hormonesjasmonic acid and ethylene.

Rhizobacteria-mediated ISR is not based on the inductionof changes in the biosynthesis of either JA or ethylene.ISR-expressing plants have the capacity to convert 1-aminocyclopropane-1-carboxylate (ACC) to ethyleneproviding a greater potential to produce ethylene uponpathogen attack.

Fluorescent pseudomonads are also known to producesalicylic acid, which acts as local and systemic signalmolecules in inducing resistance in plants

Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET)are involved in the regulation of basal resistance againstdifferent pathogens. These three signals play importantroles in induced resistance as well. SA is a key regulator ofpathogen-induced systemic acquired resistance (SAR)whereas JA and ET are required for rhizobacteria-mediatedinduced systemic resistance (ISR).

Both types of induced resistance are effective against abroad spectrum of pathogens. Comparison of theeffectiveness of SAR and ISR using a fungal, a bacterial, anda viral pathogen in non-induced Arabidopsis plants, thesepathogens are primarily resisted through either SA-dependent basal resistance (Perenospora parasitica andTurnip crinkle virus (TCV)), JA/ET-dependent basalresistant responses (Alternaria barssicicola ), or acombination of SA-JA-, and ET-dependent defenses(Xanthomonas campestris pv. armoraciae).

Activation of ISR resulted in a significant level ofprotection against Alternaria brassicicola , whereas SARwas ineffective against this pathogen. Conversely,activation of SAR resulted in a high level of protectionagainst Phytophthora parasitica and TCV, whereas ISRconferred only weak and no protection against P. parasiticaand TCV, respectively.

Induction of SAR and ISR was equally effective against X.campestris pv. armoraciae. These results indicate that SARis effective against pathogens that non-induced plants areresisted through SA-dependent defenses, where as ISR iseffective against pathogens in non-induced plants andresisted through JA/ET-dependent defenses.

This suggests that SAR and ISR constitute a reinforcementof extant SA- or JA/ET-dependent basal defense responses,respectively.

Serratia marcescens 90-166 mediates induced systemicresistance to fungal, bacterial, and viral pathogens byproducing salicylic acid (SA), using the salicylateresponsive reporter plasmid pUTK21. High-pressureliquid chromatography analysis of culture extractsconfirmed the production of SA in broth culture.

Mini-Tn5phoA mutants, which did not producedetectable amounts of SA, retained ISR activity incucumber against the fungus Colletotrichumorbiculare. Strain 90-166 induced disease resistance toP. syringae pv. Tabaci in wild-type Xanthi-nc andtransgenic NahG-10 tobacco expressing salicylatehydroxylase. Results of the study indicate that SAproduced by 90-166 is not the primary bacterialdeterminant of ISR and the bacterial-mediated ISRsystem is affected by iron concentrations

Several genera of bacteria in cluding pseudomonadsare known to synthesize SA and SA is an intermediatein the biosynthesis of pyochelin siderophores(Ankenbauer and Cox, 1988). There are someindications that SA may be involved in bacteriallymediated ISR since Pseudomonas fluorescens strainCHAO, which provides ISR in tobacco to tobacconecrosis.

virus produces SA. However, the role of SA productionin CHAO-mediated ISR has not been reported.Leeman et al ., (1996) reported that P. fluorescensstrain WCS374, which provides ISR in radish against F.oxysporum f. sp. Raphani , produced SA in quantitiesthat were iron dose-dependent, and they suggestedthat ISR was due to bacterial SA production.

Recently, the involvement of SA produced by P.aeruginosa 7NSK2 in the induction of resistanceagainst Botrytis cinerea on Phaseolous vulgaris hasbeen reported

Root colonization of A. thaliana by the nonpathogenic,rhizosphere-colonizing bacterium P. fluorescens WCS417r hasbeen shown to elicit induced systemic resistance (ISR) against P.syringae pv. Tomato (Pst).

Several ethylene-response mutants were tested and showedessentially normal symptoms of Pst infection. ISR was abolishedin the ethylene-insensitive mutant etr1-1, whereas SAR wasunaffected.

Similar results were obtained with the ethylene mutants ein2through ein7, indicating that the expression of ISR requires thecomplete signal-transduction pathway of ethylene known so far.The induction of ISR by WCS417r was not accompanied byincreased of ethylene production in roots or leaves, and neitherby increases in the expression of the genes encoding the ethylenebiosynthetic enzymes 1-aminocyclopropane-1-carboxylic (ACC)synthase and ACC oxidase.

The Etr1 mutant, displaying ethylene insensitivity in the rootsonly, did not express ISR upon application of WCS417r to theroots, but did exhibit ISR when the inducing bacteria wereinfiltrated into the leaves.

These results demonstrate that, for the induction of ISR,ethylene responsiveness is required at the site of application ofinducing rhizobacteria.

The Bacillus amyloliquefaciens EXTN-1 treatedtobacco plants

showed augmented, rapid transcript accumulation ofdefense related genes including PR-1a, phenylalanineammonia-lyase, and 3-hydroxy-3methylglutaryl CoAreductase (HMGR) following inoculation with Pepper

Mild Mottle Virus ( PMMoV ).

Thus, their expression is associated with thedevelopment of both local and systemic resistance. Allthese results may indicate that EXTN-1 inducessystemic resistance via salicylic acid and jasmonicacid-dependent pathways and timely recognitionfollowed by rapid counter attack against the viralinvasion is the key differences between incompatibleinteraction and compatible one

PGPR strains B. pumilus SE34 and P. fluorescens89B61, elicited systemic protection against the blighton tomato and reduced disease. Induced protectionelicited by both PGPR strains was SA-independent butethylene- and jasmonic acid-dependent.

In Arabidopsis , selected bacterial strains trigger aSA-independent but JA and ethylene dependentpathway that nevertheless, is dependent on theregulatory factor NPR1, which is also part of the SA-dependent pathway.

Two non-inducible ecotypes of Arabidopsis areimpaired in the same gene (ISR1) and have reducedsensitivity to ethylene, confirming the importance ofethylene sensitivity in ISR signaling

USE OF PGPR ON COMMERCIAL SCALE

The development of biological products based onbeneficial micro-organisms can extend the range of optionsfor maintaining the health and yield of crops. As early as1897 a “bacteriological fertilizer for the inoculation ofcereals” was marketed under the proprietary name Alinitby Farbenfabriken vorm. Friedrich Baye r & Co.” ofElberfeld, Germany, Today’s Bayer AG.

The product was based on a Bacillus species now known bythe taxonomic name Bacillus subtilis. In the mid-1990s inthe USA , B. subtilis started to be used as seed dressing,with registrations in more than seven crops and applicationto more than 2 million ha.

This was the first major commercial success in the use ofan antagonist. In Germany, FZB 24 B. subtilis has been onthe market since 1999 and is used mainly as a seeddressing for potatoes

In response to environmental and health concernsabout extended use of pesticides, there is considerableinterest in finding alternative control approaches foruse in integrated pest management strategies for cropdiseases. It seems inevitable that fewer pesticides willbe used in the future and that greater reliance will beplaced on biological technologies including the use ofmicroorganisms as biocontrol agents.

However, microorganisms as biocontrol agentstypically have a relatively narrow spectrum of activitycompared with synthetic pesticides and often exhibitinconsistent performance in practical agriculture,resulting in limited commercial use of biocontrolapproaches for suppression of plant pathogens

Commercial development has already beenaccomplished with two products marketed as Kodiakand Epic (Gustafson inc.), in which two differentBacillus subtilis biocontrol strains were combined witha fungicide (Carboxin-PCNB-metalaxyl) for use againstsoil borne diseases.

During the 1996 season, approximately 5 million ha ofcrops were treated with these products, targetingdiseases of roots caused by Rhizoctonia solani andFusarium spp. plus promoting root mass and plantvigor through hormone-like responses and diseasecontrol.

Many root-colonizing bacteria are known to promote plantgrowth by producing gibberellins, cytokinins and indoleacetic acid and hence are called as PGPR.

The application of five commercial chitosan-basedformulations of carefully chosen PGPR developed atAuburn University, USA has previously showndemonstrable increase in the growth of nursery-raisedplants such as cucumber, pepper and tomato amongothers.

Later, seedlings of three indica rice cultivars, IR24, IP50and Jyothi raised in rice field soil amended with each of theformulations in a 1:40 (formulation: soil) ratio have shownsignificant two -fold increase in root and shoot length, andgrain yield. The observations do suggest that application ofsuch commercial bacterial formulations can serve asmicrobial inoculants for the improvement of rice growth

INTEGRATION AND MIXTURES OF PGPR

In nature biocontrol results from mixtures of antagonists,rather from high populations of a single antagonist.Moreover, mixtures of antagonists are considered toaccount for protection of disease-suppressive soils.

Consequently, application of a mixture of introducedbiocontrol agents would more closely mimic the naturalsituation and may broaden the spectrum, enhance theefficacy and reliability of biocontrol.

Strategies for forming mixtures of biocontrol agents couldbe envisioned including mixtures of organisms withdifferential plant colonization patterns; biocontrol agentsthat control different pathogens; antagonists with differentmechanisms of disease suppression; taxonomicallydifferent organisms and antagonists with differentoptimum temperature, pH and moisture conditions forplant colonization

Combination of various mechanisms of biocontrol isuseful in achieving the goal without geneticengineering (Janisiewicz , 1996). PGPR strains INR 7 (Bacillus pumilus ). GBO3 (Bacillus subtilis ), and ME1( Curtobacterium flccumfaciens ) were tested aloneand in combinations for biocontrol againstColletotrichum orbiculare (causing anthracnose),Pseudomonas syringae pv. Lachrymans (causingangular leaf spot), and Erwinia tracheiphila (causingcucurbit wilt disease). Greater suppression andenhanced consistency was observed against multiplecucumber pathogens using strains mixture (Raupachand Kloepper, 1998). Studies on combinations ofbiocontrol agents for plant disease control haveincluded mixtures of fungi, mixtures of fungi andbacteria and mixtures of bacteria .

Combinations of a strain of Trichoderma koningii withdifferent Pseudomonas spp. isolates provided greate rsuppression of take-all disease than either the fungus orthe bacterium alone.

Increased suppression of Fusarium wilt of carnation wasobserved by combining P. putida WCS358 with non-pathogenic Fusarium oxysporum Fo47.

The enhanced disease suppression may be due tosiderophore-mediated competition for iron by WCS358,which makes the pathogenic F. oxysporum strain moresensitive to competition for glucose by the non-pathogenicstrain Fo47.

Furthermore, strains of nonpathogenic Verticillium lecanii,Acremonium rutilum or Fusarium oxysporum with thefluorescent Pseudomonas spp. strains WCS358, WCS374 orWCS417 resulted in significantly better suppression ofFusarium wilt of radish compared to the single organism

BIOPESTICIDE PRODUCTION

BIOTECHNOLOGY – Biopesticide Production -Nasrine Moazami